May 8, 2014
Genetically Altering E. Coli With New Semi-Synthetic DNA Base Pairs
Lawrence LeBlond for redOrbit.com - Your Universe Online
It is well-known that all life on Earth is made up of two base pairs, known as A-T (adenine–thymine) and C-G (cytosine–guanine), which form the standard double helix DNA strand.
"Life on Earth in all its diversity is encoded by only two pairs of DNA bases, A-T and C-G, and what we've made is an organism that stably contains those two plus a third, unnatural pair of bases," TSRI Associate Professor Floyd E. Romesberg, lead author of the study, said in a statement. "This shows that other solutions to storing information are possible and, of course, takes us closer to an expanded-DNA biology that will have many exciting applications—from new medicines to new kinds of nanotechnology."
Publishing a study on this achievement in the journal Nature, Romesberg and colleagues have been working on finding pairs of molecules that could serve as new, functional DNA bases since the 1990s. As part of their work, they also looked for new functional base pairs that could also code for proteins and organisms that have never existed before.
This work was definitely no easy task, the team admitted. They knew that finding any functional new pair of DNA bases would need to bind with an affinity comparable to that of the natural A-T and C-G base pairs. New bases would also have to line up in a stable fashion alongside natural bases in the double helix.
Much like a zipper, they would have to “unzip and re-zip smoothly when worked on by natural polymerase enzymes during DNA replication and transcription into RNA. And somehow these nucleoside interlopers would have to avoid being attacked and removed by natural DNA-repair mechanisms,” the team wrote.
Despite the challenges faced, Romesberg and his colleagues, by 2008, had made big strides toward achieving their goal.
“In a study published that year, they identified sets of nucleoside molecules that can hook up across a double-strand of DNA almost as snugly as natural base pairs and showed that DNA containing these unnatural base pairs can replicate in the presence of the right enzymes.”
“In a study that came out the following year, the researchers were able to find enzymes that transcribe this semi-synthetic DNA into RNA.”
However, the work in the 2009 study was conducted in the simplified milieu of a test tube.
"These unnatural base pairs have worked beautifully in vitro, but the big challenge has been to get them working in the much more complex environment of a living cell," said Denis A. Malyshev, a member of the Romesberg laboratory who was lead author of the new report.
For the new study, the researchers injected the common E. coli bacterium with a stretch of circular DNA known as plasmid. This plasmid DNA contained both natural DNA bases along with the best-performing unnatural base pair Romesberg’s laboratory could produce, two molecules known as d5SICS and dNaM. Their goal was to get the E. coli cells to replicate the semi-synthetic DNA as normally as possible.
The team trod carefully as the the nature of their work could have raised some eyebrows.
“The greatest hurdle may be reassuring to those who fear the uncontrolled release of a new life form: the molecular building blocks for d5SICS and dNaM are not naturally in cells,” they wrote.
To get the E. coli to replicate the unnatural DNA bases, the team had to supply the molecular building blocks artificially by adding them to the fluid solution outside the cell. To get these building blocks (nucleoside triphosphates) into the cells, they had to find special triphosphate transporter molecules – they found one made by a species of microalgae that was good enough at importing the unnatural triphosphates.
"That was a big breakthrough for us—an enabling breakthrough," said Malyshev in a statement.
Though it took another entire year to complete the project, the team was met with no other hurdles as large as this one. Surprisingly, the team found that the semi-synthetic plasmid replicated with reasonable speed and accuracy, did not hamper the E. coli cells too greatly, and showed no sign of losing its unnatural base pairs to DNA repair mechanisms.
"When we stopped the flow of the unnatural triphosphate building blocks into the cells, the replacement of d5SICS–dNaM with natural base pairs was very nicely correlated with the cell replication itself—there didn't seem to be other factors excising the unnatural base pairs from the DNA," Malyshev said. "An important thing to note is that these two breakthroughs also provide control over the system. Our new bases can only get into the cell if we turn on the 'base transporter' protein. Without this transporter or when new bases are not provided, the cell will revert back to A, T, G, C, and the d5SICS and dNaM will disappear from the genome."
John Ward, a professor of molecular microbiology at University College London, found significance in TSRI’s development.
"It's definitely interesting. It's showing proof of principle and demonstating that the E.coli repair processes do not reject artifical base pairs. It could eventually allow two parallel systems to work together in the body without competing for resources to make new bioproducts," Ward, who was not involved in the study, said in an interview with The Telegraph’s Sarah Knapton.
The team said the next step will be to demonstrate the in-cell transcription of the new expanded-alphabet DNA into the RNA that feeds the protein-making machinery of cells.
"In principle, we could encode new proteins made from new, unnatural amino acids—which would give us greater power than ever to tailor protein therapeutics and diagnostics and laboratory reagents to have desired functions," Romesberg said. "Other applications, such as nanomaterials, are also possible."
Although this is promising new research for the fields of medicine and drug development, it is likely to raise ethical and safety concerns, noted Arthur Caplan, head of the division of bioethics at NYU Langone Medical School.
“Adding alphabetic letters to the genomic code will raise eyebrows about making novel life forms, but I really don’t believe the ‘playing god’ objection has much traction,” Caplan said in an interview with BusinessWeek’s Angela Zimm. “There are many potential benefits, if they can control it.”
More concerning are inadequate guidelines to ensure safety and preventing the use of techniques by those outside responsible spheres of universities and research institutes, noted Caplan.
“We still don’t have good guidelines about releasing genetically altered microbes into the environment, into humans,” he added. “We’re not there yet with good rules. But we do need some standards and guidelines in place internationally.”